1. Field of the Invention
This invention relates generally to the stabilization/preservation of biological agents in glassy matrices and more particularly to the use of plasticizers to improve the stability of proteins in hydrophilic glasses.
2. Description of the Related Art
Recent advances in the field of biotechnology have led to the production of formulations containing proteins such as enzymes, peptides and many other biological agents suitable for use as pharmaceuticals, veterinarian preparations and foods. Many of these formulations are in the form of aqueous preparations which are unstable and must be dried to preserve the efficacy of the biological agent. A protective agent is frequently required to prevent any deleterious effects as a result of the drying procedure.
It has long been known that proteins and other biological structures can be stored in a dry state while retaining some or all of their functionality. Some plants and simple animals can survive in a dry, dormant state for extended periods of time, and function normally when rehydrated. Well over a decade ago, it became clear that carbohydrate glass plays a central role in anhydrous preservation of biological agents in nature,1,2 but there is still a significant amount of uncertainty as to the exact mechanisms that are important in biopreservation.
There are probably two major classes of stabilization mechanisms. One class, kinetic stabilization, is brought about by the slow dynamics in a glass. The glass is thought to have the ability to “form fit” the protein, and impart to some degree its dynamics on the protein, and to retard the diffusion of potentially harmful, external species. There is circumstantial evidence for this mechanism, inasmuch as the crystalline phase of a material will not provide stabilization to proteins, while the glassy form of the same material will provide good stabilization.3,4 The other major mechanistic class, thermodynamic stabilization, is often viewed as being mediated through the ability of the carbohydrate to “replace” water at the protein surface. Tanaka et al.5 showed that catalase denaturation upon freeze-drying was minimized when there was at least enough glass-former to occupy the hydrophilic sites on the protein. Cleland et al.6 reported supporting results for minimization of soluble aggregation formation of rhGH (recombinant human growth hormone) in lyophilized form. Carpenter et al.7 performed similar experiments on a monoclonal antibody, and found that in order to maximally stabilize the antibody against several stress modes, a mole ratio of lyoprotectant to protein was needed that was twice that reported as required to protect against aggregation or denaturation on drying. Other components in the formulation, such as ionic species8,9,10 and surfactants may also contribute to the thermodynamic stabilization of the protein in the glass.
Typical materials for the formulation of lyoprotective glasses have been generally limited to single sugars and sugar alcohols,12,13,14,15 polymeric sugars such as inulin,16 ficoll and dextran,17,18 synthetic polymers such as dextran DEA, dextran sulfate, polyethylene glycol, polyvinyl pyrrolidone, polyacrylamide, or polyethyleneimine,19,20 and some amino acids.21 Materials such as those mentioned above are referred to as “standard” glassforming materials because they are in more-or-less common use.
Various mixtures of the standard glassforming materials have also been used in formulations for stabilization of proteins, such as polymers and small carbohydrates.22,23,24,25 These mixtures can yield materials with desirable physical characteristics, such as increased mechanical toughness, increased glass transition temperature (Tg), or decreased probability of crystallization. The desirability of these characteristics is often related to ease of processing, but some of these mixtures are also reported to yield increased lifetime of stabilized protein in the glass.23,24 Amino acids or crystallizing alcohols used as additives in carbohydrate glasses comprise another class of mixtures of standard materials. Additives in this class have very high melting temperatures (Tm>100° C.), and typically have Tg values within 20° C. to 30° C. of room temperature. The wide temperature range between Tm and Tg makes these materials poor glassformers, and predisposes them to crystallization rather than glass-formation. While they are known to be mild plasticizers to the glass,26,27 they are typically used because of their propensity to crystallize. These compounds are often induced to crystallize during the freezing step, thus they become a support against structural collapse during drying.28 Upon crystallization they may also form micro-chambers, inhibiting phase separation.29 While they do impart desirable physical characteristics to the formulation, the presence of crystalline mannitol or glycine in an otherwise glassy formulation has been shown to be destabilizing to several proteins.30,4 
One recent and important improvement to the standard-materials-only approach is the addition of borate ion.31 The action of borate ion is very different than the action of salts typically added to glassy biopreservation formulations. Borate is known to function as a cross-linking agent for OH groups (e.g. on the sugars), and its addition to a carbohydrate glass increases the Tg of the mixture by as much as 80° C.32 Thus, it acts as a powerful anti-plasticizing agent, whereas the presence of monovalent33,34 and divalent cations10 does not significantly alter Tg of the glass by comparison.
It is important to note that essentially all of the “standard” glassforming materials in use for preserving biological agents in a dry state have glass transitions above or near room temperature. We are aware of two exceptions: surfactants, which are employed as a protection against unfolding during freezing,11 and t-butyl alcohol (TBA), which was used in freeze-drying as a facilitating agent for sublimation of ice crystals.35 The authors of that report indicated that the presence of TBA did not change the collapse temperature of the freeze-dried cake. Thus, the TBA did not act as a plasticizer, probably because it sublimed off with the water. Other than these exceptions, materials with Tg far below room temperature are not typically used to enhance the biopreserving efficacy of a lyoprotective glass. This is not surprising, as these materials will typically be strong plasticizers of the glass (reduce its Tg), and this is typically viewed as undesirable.36 It is sometimes observed37 and commonly asserted38,39 that a higher host Tg will lead to better preservation of biological agents at a given storage temperature. One might conclude from such observations and assertions that plasticization is always deleterious to the bio-stabilizing capacity, and in fact, statements have been made in the literature with that underlying assumption.40 
Small-molecule hydrophilic solutes such as glycerol, propylene glycol, and dimethyl sulfoxide (DMSO) are commonly used to stabilize proteins in solution against cryogenic stress. However, being liquids at room temperature, and possessing Tg values far below room temperature, they are strong plasticizers, and for the reasons mentioned above are typically avoided in lyoprotective glass formulations. There is one study where they have been evaluated in glasses for preservation of fungus spores. In the study, addition of ˜8 wt % glycerol or DMSO to the glass formulation was tested for fungus preservation.41 The authors in this study observed a negative effect on the fungus spore stability caused by plasticizers.
There are two studies wherein glycerol was present in the biopreservation formulation somewhere in the range of 20 wt % to 22 wt %, and originated from the solution in which the enzyme was supplied.42,43 In both of these cases, the formulation tested was not glassy. One of these reports included reasonable levels of protein stabilization, although those results were not quantitative.43 Several studies have shown that proteins can be stabilized to some extent in the presence of glycerol alone, or solutions of glycerol, at temperatures well above the glass transition temperature of the solution.44,45,46 This is thought to occur by an indirect mechanism that involves hydration water.47 
In a series of similar single-component glasses, the glasses with lower Tg will have slower dynamics at Tg.48 This effect is due, at least in part, to a reduced upper length scale of motions that are relevant to the glassy dynamics. In spite of cautions to the contrary in the literature, the present inventors reasoned that the addition of a plasticizer might slow the dynamics within the glass, and improve stability of preserved proteins, provided that the small molecule is dynamically linked to the larger glass-forming molecule. This proviso is non-trivial, as individual components in mixtures of materials with widely differing Tg values are known to exhibit uncoupled dynamics.49 
Despite the progress which has been made in understanding the underlying mechanisms of protective agents such as glassy matrices in freeze-drying of biological agents such as enzymes, there remains a need to develop methods and compositions which improve the long-term stability of dried biological agents.